Resistance to auxinic herbicides

ABSTRACT

The invention provides methods of identifying herbicidal auxins. The invention further provides auxin-herbicide-resistant plants and genes conferring auxin-herbicide resistance. This invention also provides a method of identifying other proteins that bind picolinate auxins from additional plant species. The invention further provides a method to identify the molecular binding site for picolinate auxins. The invention also includes the use of the picolinate herbicidal auxin target site proteins, and methods of discovering new compounds with herbicidal or plant growth regulatory activity. The invention also includes methods for producing plants that are resistant to picolinate herbicidal auxins. Specific examples of novel proteins associated with herbicide binding include AFB5, AFB4, and SGT1b.

REFERENCE TO RELATED APPLICATIONS

This is a divisional of co-pending U.S. patent application Ser. No.12/875,946, filed Sep. 3, 2010, which in turn claims the benefit of U.S.patent application Ser. No. 11/686,844, filed Mar. 15, 2007, and U.S.Provisional Application No. 60/783,015, filed Mar. 15, 2006, all ofwhich are incorporated herein in their entirety.

FIELD OF THE DISCLOSURE

The present invention relates to the field of plant biology andspecifically to methods of identifying herbicidal auxins and herbicideresistant plants, and to nucleotide sequences conferring herbicideresistance to plants, particularly resistance to picolinate class ofauxinic herbicides.

BACKGROUND OF THE DISCLOSURE

The use of herbicides for weed control is an extremely valuableagricultural practice to protect the yield of crop plants and to managethe growth of vegetation in pastures and other sites. However the use ofmany herbicides is becoming problematic through the advent of fieldresistance and also from increased toxicological and environmentalconcerns associated with certain pesticidal chemistries and modes ofaction. Thus there is a continuing need for new herbicidal chemistries.

There are currently a limited number of herbicidal classes that haveproven to exhibit the desired attributes for a beneficial modernherbicide such as low incidence of resistance development and lowpotential for toxicological side effects. One such class is the auxinicherbicides that include such compounds as 2,4-D and picloram. This classcan be further subdivided into compounds that contain a picolinatemoiety (e.g., picloram, aminopyralid, clopyralid), those that contain anaryloxyacetate moiety (e.g., 2,4-D, fluoroxypyr, triclopyr etc.) andothers (e.g., dicamba). All of these compounds elicit plant symptomssimilar to excessive treatment with the natural plant hormone indoleacetic acid (IAA) and induce similar physiological events, eventuallyleading to plant death. Interestingly, the widespread use of auxinicherbicides for many years has not resulted in significant fieldresistance. In addition, the auxinic mode of action is specific toplants and many of the auxinic herbicides exhibit favorableenvironmental profiles. Thus methods for the discovery of new compoundsthat act via the auxinic mode of action would be of great benefit,particularly those with increased potency, wider spectrum, optimal soilpersistency or low cost of manufacture.

Genes that confer resistance to certain herbicides have found utility inthe development of herbicide-tolerant crops by transgenic or mutagenicselection methods. Many auxinic herbicides have broad spectrumherbicidal activity but their use is limited by the fact that thedesired crop or pasture component species is sensitive to the herbicide.This is particularly the case for the picolinate class of auxinicherbicides. Thus there is an unfulfilled need for herbicide-tolerancemechanisms that would be applicable to the picolinate class of auxinicherbicides.

SUMMARY OF THE DISCLOSURE

The invention in various embodiments provides methods of identifyingherbicidal auxins (reporter assays, co-crystal structures of picolinateauxins and other chemistries that interact with AFB 4, AFB5 or SGT1b.Also provided are auxin herbicide resistant plants and genes conferringauxin-herbicide resistance. This disclosure also provides a method ofidentifying other proteins that bind picolinate auxins from additionalplant species. These proteins may be identified by sequence similaritywith AFB5 or SGT1b, the picolinate auxin target proteins disclosed here.

The invention in various embodiments further provides a method toidentify the molecular binding site for picolinate auxins on AFB4 andAFB5 by performing domain swap experiments between AFB5 and the closelyrelated F-box protein TIR1. Both AFB5 and TIR1 participate in auxinsignal transduction; however TIR1 does not significantly interact withcertain picolinate auxins. The binding site for the picolinate auxinsmay be identified by combining different portions of AFB5 and TIR1 andassaying for picolinate auxin binding to the chimeric protein.

As discussed in more detail below, AFB4 is another AFB protein of thesubject invention. AFB4 shares about 80% amino acid identity with AFB5.AFB4 and AFB5 homologs are distinct in various ways (at the sequencelevel and in light of their unique properties, for example) from TIR1and previously known AFB proteins such as AFB 1, AFB2, and AFB3. AFB1 isabout 45% identical to AFB4 and about 47% identical to AFB5. AFB3 isabout 48% identical to AFB4 and about 49% identical to AFB5.

The disclosure also includes the use of the picolinate herbicidal auxintarget site proteins in discovering new compounds with herbicidal orplant growth regulatory activity. In one embodiment, the proteins can beused in biochemical assays to identify compounds that effectivelyinhibit ligand-binding to the target protein or inhibit auxinfunctionality. In another embodiment the proteins may be crystallized inthe presence or absence of the herbicide and the molecular structure ofthe proteins determined by X-ray crystallography. This structuralinformation can be used to design or search for new chemical structuresthat effectively interact with the target protein.

The disclosure also includes methods for producing plants that areresistant to picolinate herbicidal auxins. This may be accomplishedthrough the generation of transgenic plants with gene constructs thatinhibit the expression of the target protein or by screening for plantsthat are resistant to the picolinate auxin and using molecularinformation about the gene encoding the target protein to identify themutation responsible for the auxin resistance or in plant breedingexperiments that may enable the movement of the resistance gene intoother plant varieties.

Picolinate auxinic herbicides exert their herbicidal effects throughspecific interaction with certain proteins and these proteins do notinteract significantly with other auxinic herbicides, such as 2,4-D. Onesuch protein is AFB5. AFB5 shows sequence similarity to a reported auxinreceptor, the F-box protein, TIR1, that has been shown to mediate theinteraction of the natural hormone IAA (and the herbicide 2,4-D) toinitiate the auxin signal transduction cascade (Dharmasiri et al.,Nature 435:441-445, 2005; Kepinski and Leyser, Nature 435:446-451,2005). TIR1 acts as the recognition component of an SCF E3-ubiquitinligase complex that directs the proteosomal degradation ofauxin-responsive transcriptional regulators. The advantage of thepresent invention is that AFB5 specifically mediates the interaction ofpicolinate herbicidal auxins and not IAA or 2,4-D such that loss of itsfunction confers resistance to picolinate auxins specifically andstimulation of its function leads to a herbicidal signal cascade.Another protein discovered to have similar properties is SGT1b. This isalso associated with SCF function and is involved in mediatingprotein-protein interactions. A feature of this invention is that SGT1bfunction is specifically involved in picolinate auxin action.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. Differential effects of auxins on root growth of mutantsfrom complementation Groups 1 and 2. Root lengths of Arabidopsisseedlings were measured 8 days after seeding on plates containing arange of concentrations of the test compounds and are plotted as apercentage of the root length of untreated plants in the sameexperiment. Typical root lengths for untreated controls were 21, 21 and24 mm for wild-type, Group 1, and Group 2 plants respectively. Thesymbols used for wild type, R009 from complementation Group 1 and R051from complementation Group 2 are denoted in FIG. 1A. (FIG. 1A) DAS534,(FIG. 1B) 2,4-D, (FIG. 1C) picloram, (FIG. 1D) IAA.

FIGS. 2A-2J. DAS534-resistant mutants and complementation with AFB5 andSGT1b. (FIG. 2A) to (FIG. 2E) untreated seedlings, (FIG. 2F) to (FIG.2J) seedlings grown in the presence of 5 nM DAS534. The primary visibleeffects at this sublethal concentration of auxin are loss ofgravitropism, lack of cotyledon decurvature and increased hypocotylelongation (FIG. 2F). Mutants R127 and R051 are resistant to this effect(FIG. 2G) and (FIG. 2I), whereas mutants transformed with thecorresponding wild-type gene regain the response, (FIG. 2H) and (FIG.2J). The R127CsVMV:AFB5 line appears to have an increased responserelative to Col-0 (FIG. 2H).

FIGS. 3A-3B. Map-based cloning of the resistance mutations in R009 andR051. The horizontal line represents the chromosome, the verticalhatched lines show the position of the markers used in fine mappingexperiments. (FIG. 3A) mapping of R009, (FIG. 3B) mapping of R051.

FIGS. 4A-4C. Comparison of the amino acid sequences of six TIR1-relatedF-box proteins from Arabidopsis and COIL Sequences were aligned usingCLUSTAL within the Vector NTI suite. The F-box domain is denoted by thehatched line and the N-terminal extension unique to AFB5 and AFB4 by thesolid line. Identical residues in all five AFBs are shaded black,residues that have conservative substitutions in one or more AFBs areshaded gray. The sites of the three mutant alleles of AFB5 are shown initalics, and those previously described for TIR1 are shown withunderlining (Ruegger et al., Genes Dev 12: 198-207, 1998; Alonso et al.,Proc Natl Acad Sci USA. 100(5):2992-2997, 2003). AFB1 is gi|18412177,AFB2 is gi|18405102, AFB3 is gi|18391439, AFB4 is gi142573021, AFB5 isgi|18423092, TIR1 is gi|18412567 and COI1 is gi|18405209.

FIGS. 5A-5C. Comparison of the effect of DAS534 and 2,4-D on previouslycharacterized mutants. Measurements were performed as described in FIG.2. Typical root lengths for untreated controls were 20, 39 and 31 mm fortir1-1, axr2-1 and axr1-3 plants respectively. (FIG. 5A) Effect ofDAS534 on edm1 and R051. The deletion mutant line edm1 lacks seven genes(At4g11220 through At4g11280) including SGT1b (Tor et al., Plant Cell14: 993-1003, 2002). Effect of DAS534 (FIG. 5B) and 2,4-D (FIG. 5C) onaxr1-3, axr2-1 and tir1-1 compared with R009 and R051.

FIGS. 6A-6B. Mutation sites in the SGT1b (At4g11260) gene that conferresistance to DAS534. (FIG. 6A) The exons of SGT1b are shown as boxedregions. The mutations in R107, R051 and R118 occur at the 3′ splicingsites of introns 6 and 7 of the gene. (FIG. 6B) The nucleotide sequencesof the 3′ intron-exon boundaries for introns 6 and exon 7 and for intron7 and exon 8 are shown. The intron portion is shaded gray and themutation sites are arrowed. The complete nucleotide sequences ofAt4g11260 can be found in gi130698542 as the complement of sequences6851273 through 6853848.

FIG. 7 illustrates a phylogenetic analysis showing that AFB5 (and fourrelated AFBs, including AFB4) are distinct from previously characterizedTIR1 (and six related proteins, including AFB1, AFB2, and AFB3).AtAFB14g03190 is gi|18412177, AtAFB2 3g26810 is gi|18405102, AtAFB31g12820 is gi|18391439, AtAFB4 4g24390 is gi142573021, AtAFB5 At5g49980is gi|18423092, AtTIR1At3g62980 is gi|18412567, Os XP 493919 isgi/50949123, Os CAD40545 is gi121740736, Os NP 912552 is gi134902412, OsXP 507533 is gi151979370 and Pt AAK16647 is gi|13249030.

FIGS. 8A-8C show a comparison of the effect of a foliar application of200 g/ha picloram (FIG. 8A), 200 g/ha aminopyralid (FIG. 8B) or 50 g/ha2,4-D (FIG. 8C) on wild type Col-0 and mutant R009 Arabidopsis plants.Pictures were taken 12 days after treatment of 17-day old plants.

BRIEF DESCRIPTION OF THE SEQUENCES

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile in the form of the file named “Sequence.txt,” which was created onNov. 15, 2011, and is 85,301 bytes, which is incorporated by referenceherein.

In the accompanying sequence listing:

SEQ ID NO: 1 provides the nucleic acid sequence for the Arabidopsis geneidentified herein as AFB5 and being a picolinate auxin receptor andassociated with resistance to this class of herbicides(gi|30695799:135-1994 Arabidopsis thaliana transport inhibitor responseprotein, putative (At5g49980) mRNA, complete cds).

SEQ ID NO: 2 provides the AFB5 protein sequence encoded by SEQ ID NO: 1(gi|18423092|ref|NP_(—)568718.1| transport inhibitor response protein,putative [Arabidopsis thaliana]).

SEQ ID NO: 3 provides the nucleic acid sequence for the Arabidopsis geneidentified herein as SGT1b and being associated with picolinate auxinbinding and resistance (gi|17017309|gb|AF439976.11 Arabidopsis thalianaSGT1b (SGT1b) mRNA, complete cds).

SEQ ID NO: 4 provides the SGT1b protein sequence encoded by SEQ ID NO: 3(gi|17017310|gb|AAL33612.1|AF439976_(—)1 SGT1b [Arabidopsis thaliana]).

SEQ ID NO: 5 provides the nucleic acid sequence for the Arabidopsis geneidentified herein as AFB4 (gi|42573020|ref|NM_(—)202878.1|Arabidopsisthaliana ubiquitin-protein ligase AT4G24390 transcript variantAT4G24390.2 mRNA, complete cds).

SEQ ID NO: 6 provides the AFB4 protein sequence encoded by SEQ ID NO: 5(gi|42573021|ref|NP_(—)974607.1|ubiquitin-protein ligase [Arabidopsisthaliana]).

SEQ ID NO: 7 provides the nucleic acid sequence of an AFB5 homolog fromOryza sativa (rice) (GenBank entry gi:34902411).(gi|34902411|ref|NM_(—)187663.11Oryza sativa (japonica cultivar-group),predicted mRNA).

SEQ ID NO: 8 provides the protein sequence encoded by SEQ ID NO: 7.(gi|34902412|ref|NP_(—)912552.1|Putative F-box containing protein TIR1[Oryza sativa (japonica cultivar-group)]).

SEQ ID NO: 9 provides the nucleic acid sequence of another AFB5 homologfrom Oryza sativa (rice) (GenBank entry gi:51979369).(gi|51979369|ref|XM_(—)507533.1| PREDICTED Oryza sativa (japonicacultivar-group), OJ1175_B01.8-1 mRNA).

SEQ ID NO: 10 provides the protein sequence encoded by SEQ ID NO: 9.(gi|51979370|ref|XP_(—)507533.1| PREDICTED OJ1175_B01.8-1 gene product[Oryza sativa (japonica cultivar-group)]).

SEQ ID NO: 11 provides the nucleic acid sequence of an AFB5 homolog fromPopulus (gi|13249029|gb|AF139835.1|AF139835 Populus tremula×Populustremuloides F-box containing protein TIR1 (TIR1) mRNA, complete cds).

SEQ ID NO: 12 provides the protein sequence encoded by SEQ ID NO: 11(gi|13249030|gb|AAK16647.1|AF139835_(—)1 F-box containing protein TIR1[Populus tremula×Populus tremuloides]).

SEQ ID NO: 13 provides the amino acid sequence of the myc epitope.

DETAILED DESCRIPTION OF THE INVENTION Terms

Unless otherwise indicated, all technical and scientific terms usedherein have the same meaning as they would to one skilled in the art ofthe present invention. Practitioners are particularly directed toSambrook et al. (Molecular Cloning, Cold Spring Harbor, 1989) andAusubel et al., (Current Protocols in Molecular Biology, 1993), fordefinitions and terms of the art. It is to be understood that thisinvention is not limited to the particular methodology, protocols, andreagents described, as these may vary.

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

As used herein, the term “herbicide” refers to a chemical compoundemployed to kill or suppress the growth of plants, plant cells andtissues or to prevent or suppress the germination and growth of plantseeds.

As used herein, the term “vector” refers to a nucleic acid constructdesigned for transfer between different host cells. An “expressionvector” refers to a vector that has the ability to incorporate andexpress heterologous DNA fragments in a foreign cell. Many prokaryoticand eukaryotic expression vectors are commercially available. Selectionof appropriate expression vectors is within the knowledge of thosehaving skill in the art.

A “heterologous” nucleic acid construct or sequence has a portion of thesequence that is not native to the plant cell in which it is expressed.Heterologous, with respect to a control sequence refers to a controlsequence (i.e. promoter or enhancer) that does not function in nature toregulate the same gene the expression of which it is currentlyregulating. Generally, heterologous nucleic acid sequences are notendogenous to the cell or part of the genome in which they are present,and have been added to the cell, by infection, transfection,microinjection, electroporation, or the like. A “heterologous” nucleicacid construct may contain a control sequence/DNA coding sequencecombination that is the same as, or different from a controlsequence/DNA coding sequence combination found in the native plant.

As used herein, the term “gene” means the segment of DNA involved inproducing a polypeptide chain, which may or may not include regionspreceding and following the coding region, e.g. 5′ untranslated (5′ UTR)or “leader” sequences and 3′ UTR or “trailer” sequences, as well asintervening sequences (introns) between individual coding segments(exons) and non-transcribed regulatory sequence.

As used herein, “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid sequence or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention.

As used herein, the term “gene expression” refers to the process bywhich a polypeptide is produced based on the nucleic acid sequence of agene. The process includes both transcription and translation;accordingly, “expression” may refer to either a polynucleotide orpolypeptide sequence, or both. Sometimes, expression of a polynucleotidesequence will not lead to protein translation. “Over-expression” refersto increased expression of a polynucleotide and/or polypeptide sequencerelative to its expression in a wild-type (or other reference [e.g.,non-transgenic]) plant and may relate to a naturally-occurring ornon-naturally occurring sequence. “Ectopic expression” refers toexpression at a time, place, and/or increased level that does notnaturally occur in the non-altered or wild-type plant.“Under-expression” refers to decreased expression of a polynucleotideand/or polypeptide sequence, generally of an endogenous gene, relativeto its expression in a wild-type plant. The terms “mis-expression” and“altered expression” encompass over-expression, under-expression, andectopic expression.

The term “introduced” in the context of inserting a nucleic acidsequence into a cell, means “transfection”, or “transformation” or“transduction” and includes reference to the incorporation of a nucleicacid sequence into a eukaryotic or prokaryotic cell where the nucleicacid sequence may be incorporated into the genome of the cell (forexample, chromosome, plasmid, plastid, or mitochondrial DNA), convertedinto an autonomous replicon, or transiently expressed (for example,transfected mRNA).

As used herein, a “plant cell” refers to any cell derived from a plant,including cells from undifferentiated tissue (e.g., callus) as well asplant seeds, pollen, propagules and embryos.

As used herein, the terms “native” and “wild-type” relative to a givenplant trait or phenotype refers to the form in which that trait orphenotype is found in the same variety of plant in nature.

As used herein, the term “modified” regarding a plant trait, refers to achange in the phenotype of a transgenic plant relative to the similarnon-transgenic plant. An “interesting phenotype (trait)” with referenceto a transgenic plant refers to an observable or measurable phenotypedemonstrated by a T1 and/or subsequent generation plant, which is notdisplayed by the corresponding non-transgenic (i.e., a genotypicallysimilar plant that has been raised or assayed under similar conditions).An interesting phenotype may represent an improvement in the plant ormay provide a means to produce improvements in other plants. An“improvement” is a feature that may enhance the utility of a plantspecies or variety by providing the plant with a unique and/or novelquality.

As used herein, a “mutant” polynucleotide sequence or gene differs fromthe corresponding wild type polynucleotide sequence or gene either interms of sequence or expression, where the difference contributes to amodified plant phenotype or trait. Relative to a plant or plant line,the term “mutant” refers to a plant or plant line which has a modifiedplant phenotype or trait, where the modified phenotype or trait isassociated with the modified expression of a wild type polynucleotidesequence or gene.

As used herein, the term “T1” refers to the generation of plants fromthe seed of T0 plants. The T1 generation is the first set of transformedplants that can be selected by application of a selection agent, e.g.,an antibiotic or herbicide, for which the transgenic plant contains thecorresponding resistance gene. The term “T2” refers to the generation ofplants by self-fertilization of the flowers of T1 plants, previouslyselected as being transgenic. T3 plants are generated from T2 plants,etc. As used herein, the “direct progeny” of a given plant derives fromthe seed (or, sometimes, other tissue) of that plant and is in theimmediately subsequent generation; for instance, for a given lineage, aT2 plant is the direct progeny of a T1 plant. The “indirect progeny” ofa given plant derives from the seed (or other tissue) of the directprogeny of that plant, or from the seed (or other tissue) of subsequentgenerations in that lineage; for instance, a T3 plant is the indirectprogeny of a T1 plant.

As used herein, the term “plant part” includes any plant organ ortissue, including, without limitation, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores. Plant cells can be obtained fromany plant organ or tissue and cultures prepared therefrom. The class ofplants which can be used in the methods of the present invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledenous anddicotyledenous angiosperm as well as gymnosperm plants.

As used herein, the term “gene silencing” refers to lack of (orreduction of) gene expression as a result of, though not limited to,effects at a genomic (DNA) level such as chromatin re-structuring, or atthe post-transcriptional level through effects on transcript stabilityor translation. Evidence suggests that RNA interference (RNAi) is amajor process involved in transcriptional and posttranscriptional genesilencing. Because RNAi exerts its effects at the transcriptional and/orpost-transcriptional level, it is believed that RNAi can be used tospecifically inhibit alternative transcripts from the same gene.

As used herein, the terms “interfering with” or “inhibiting” (expressionof a target sequence) refers to the ability of a small RNA, such as ansiRNA or a miRNA, or other molecule, to measurably reduce the expressionand/or stability of molecules carrying the target sequence. A targetsequence can include a DNA sequence, such as a gene or the promoterregion of a gene, or an RNA sequence, such as an mRNA. “Interfering withor inhibiting” expression contemplates reduction of the end-product ofthe gene or sequence, e.g., the expression or function of the encodedprotein or a protein, nucleic acid, other biomolecule, or biologicalfunction influenced by the target sequence, and thus includes reductionin the amount or longevity of the mRNA transcript or other targetsequence. In some embodiments, the small RNA or other molecule guideschromatin modifications which inhibit the expression of a targetsequence. It is understood that the phrase is relative, and does notrequire absolute inhibition (suppression) of the sequence. Thus, incertain embodiments, interfering with or inhibiting expression of atarget sequence requires that, following application of the small RNA orother molecule (such as a vector or other construct encoding one or moresmall RNAs), the sequence is expressed at least 5% less than prior toapplication, at least 10% less, at least 15% less, at least 20% less, atleast 25% less, or even more reduced. Thus, in some particularembodiments, application of a small RNA or other molecule reducesexpression of the target sequence by about 30%, about 40%, about 50%,about 60%, or more. In specific examples, where the small RNA or othermolecule is particularly effective, expression is reduced by 70%, 80%,85%, 90%, 95%, or even more.

As used herein, the term “Post-Transcriptional Gene Silencing” (PTGS)refers to a form of gene silencing in which the inhibitory mechanismoccurs after transcription. This can result in either decreasedsteady-state level of a specific RNA target or inhibition of translation(Tuschl, ChemBiochem, 2:239-245, 2001). In the literature, the terms RNAinterference (RNAi) and posttranscriptional cosuppression are often usedto indicate posttranscriptional gene silencing.

As used herein, the term “regulating gene expression” refers to theprocess of controlling the expression of a gene by increasing ordecreasing the expression, production, or activity of an agent thataffects gene expression. The agent can be a protein, such as atranscription factor, or a nucleic acid molecule, such as a miRNA or ansiRNA molecule, which when in contact with the gene or its upstreamregulatory sequences, or a mRNA encoded by the gene, either increases ordecreases gene expression.

As used herein, the term “RNA interference” (RNAi) refers to genesilencing mechanisms that involve small RNAs (including miRNA and siRNA)are frequently referred to under the broad term RNAi. Natural functionsof RNAi include protection of the genome against invasion by mobilegenetic elements such as transposons and viruses, and regulation of geneexpression.

RNA interference results in the inactivation or suppression ofexpression of a gene within an organism. RNAi can be triggered by one oftwo general routes. First, it can be triggered by direct cellulardelivery of short-interfering RNAs (siRNAs, usually ˜21 nucleotides inlength and delivered in a dsRNA duplex form with two unpairednucleotides at each 3′ end), which have sequence complementarity to aRNA that is the target for suppression. Second, RNAi can be triggered byone of several methods in which siRNAs are formed in vivo from varioustypes of designed, expressed genes. These genes typically express RNAmolecules that form intra- or inter-molecular duplexes (dsRNA) or a“hairpin” configuration which are processed by natural enzymes (DICER orDCL) to form siRNAs. In some cases, these genes express“hairpin”-forming RNA transcripts with perfect or near-perfectbase-pairing; some of the imperfect hairpin-forming transcripts yield aspecial type of small RNA, termed microRNA (miRNA). In either generalmethod, it is the siRNAs (or miRNAs) that function as “guide sequences”to direct an RNA-degrading enzyme (termed RISC) to cleave or silence thetarget RNA. In some cases, it is beneficial to integrate anRNAi-inducing gene into the genome of a transgenic organism. An examplewould be a plant that is modified to suppress a specific gene by anRNAi-inducing transgene. In most methods that are currently in practice,RNAi is triggered in transgenic plants by transgenes that express adsRNA (either intramolecular or hairpin, or intermolecular in which twotranscripts anneal to form dsRNA).

As used herein, the term “RNA silencing” is a general term that is usedto indicate RNA-based gene silencing or RNAi.

As used herein, the term “silencing agent” or “silencing molecule”,refers to a specific molecule, which can exert an influence on a cell ina sequence-specific manner to reduce or silence the expression orfunction of a target, such as a target gene or protein. Examples ofsilence agents include nucleic acid molecules such as naturallyoccurring or synthetically generated small interfering RNAs (siRNAs),naturally occurring or synthetically generated microRNAs (miRNAs),naturally occurring or synthetically generated dsRNAs, and antisensesequences (including antisense oligonucleotides, hairpin structures, andantisense expression vectors), as well as constructs that code for anyone of such molecules.

As used herein, the term “small interfering RNA” (siRNA) refers to a RNAof approximately 21-25 nucleotides that is processed from a dsRNA by aDICER enzyme (in animals) or a DCL enzyme (in plants). The initial DICERor DCL products are double-stranded, in which the two strands aretypically 21-25 nucleotides in length and contain two unpaired bases ateach 3′ end. The individual strands within the double stranded siRNAstructure are separated, and typically one of the siRNAs then areassociated with a multi-subunit complex, the RNAi-induced silencingcomplex (RISC). A typical function of the siRNA is to guide RISC to thetarget based on base-pair complementarity.

As used herein, the term “transcriptional gene silencing” (TGS) refersto a phenomenon that is triggered by the formation of dsRNA that ishomologous with gene promoter regions and sometimes coding regions. TGSresults in DNA and histone methylation and chromatin remodeling, therebycausing transcriptional inhibition rather than RNA degradation. Both TGSand PTGS depend on dsRNA, which is cleaved into small (21-25nucleotides) interfering RNAs (Eckhardt, Plant Cell, 14:1433-1436, 2002;Aufsatz et al., Proc. Natl. Acad. Sci. U.S.A., 99:16499-16506, 2002).

As used herein, “transgenic plant” includes a plant that compriseswithin its genome a heterologous polynucleotide. The heterologouspolynucleotide can be either stably integrated into the genome, or canbe extra-chromosomal. Preferably, the polynucleotide of the presentinvention is stably integrated into the genome such that thepolynucleotide is passed on to successive generations. A plant cell,tissue, organ, or plant into which the heterologous polynucleotides havebeen introduced is considered “transformed”, “transfected”, or“transgenic”. Direct and indirect progeny of transformed plants or plantcells that also contain the heterologous polynucleotide are alsoconsidered transgenic.

Various methods for the introduction of a desired polynucleotidesequence encoding the desired protein into plant cells are available andknown to those of skill in the art and include, but are not limited to:(1) physical methods such as microinjection, electroporation, andmicroprojectile mediated delivery (biolistics or gene gun technology);(2) virus mediated delivery methods; and (3) Agrobacterium-mediatedtransformation methods.

The most commonly used methods for transformation of plant cells are theAgrobacterium-mediated DNA transfer process and the biolistics ormicroprojectile bombardment mediated process (i.e., the gene gun).Typically, nuclear transformation is desired but where it is desirableto specifically transform plastids, such as chloroplasts or amyloplasts,plant plastids may be transformed utilizing a microprojectile-mediateddelivery of the desired polynucleotide.

Agrobacterium-mediated transformation is achieved through the use of agenetically engineered soil bacterium belonging to the genusAgrobacterium. A number of wild-type and disarmed strains ofAgrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti orRi plasmids can be used for gene transfer into plants. Gene transfer isdone via the transfer of a specific DNA known as “T-DNA” that can begenetically engineered to carry any desired piece of DNA into many plantspecies.

Agrobacterium-mediated genetic transformation of plants involves severalsteps. The first step, in which the virulent Agrobacterium and plantcells are first brought into contact with each other, is generallycalled “inoculation”. Following the inoculation, the Agrobacterium andplant cells/tissues are permitted to be grown together for a period ofseveral hours to several days or more under conditions suitable forgrowth and T-DNA transfer. This step is termed “co-culture”. Followingco-culture and T-DNA delivery, the plant cells are treated withbactericidal or bacteriostatic agents to kill or inhibit theAgrobacterium remaining in contact with the explant and/or in the vesselcontaining the explant. If this is done in the absence of any selectiveagents to promote preferential growth of transgenic versusnon-transgenic plant cells, then this is typically referred to as the“delay” step. If done in the presence of selective pressure favoringtransgenic plant cells, then it is referred to as a “selection” step.When a “delay” is used, it is typically followed by one or more“selection” steps.

With respect to microprojectile bombardment (U.S. Pat. No. 5,550,318(Adams et al.); U.S. Pat. No. 5,538,880 (Lundquist et. al.), U.S. Pat.No. 5,610,042 (Chang et al.); and PCT Publication WO 95/06128 (Adams etal.); each of which is specifically incorporated herein by reference inits entirety), particles are coated with nucleic acids and deliveredinto cells by a propelling force. Exemplary particles include thosecomprised of tungsten, platinum, and preferably, gold.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System(BioRad, Hercules, Calif.), which can be used to propel particles coatedwith DNA or cells through a screen, such as a stainless steel or Nytexscreen, onto a filter surface covered with monocot plant cells culturedin suspension.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species thathave been transformed by microprojectile bombardment include monocotspecies such as maize (International Publication No. WO 95/06128 (Adamset al.)), barley, wheat (U.S. Pat. No. 5,563,055 (Townsend et al.)incorporated herein by reference in its entirety), rice, oat, rye,sugarcane, and sorghum; as well as a number of dicots including tobacco,soybean (U.S. Pat. No. 5,322,783 (Tomes et al.), incorporated herein byreference in its entirety), sunflower, peanut, cotton, tomato, andlegumes in general (U.S. Pat. No. 5,563,055 (Townsend et al.)incorporated herein by reference in its entirety).

To select or score for transformed plant cells regardless oftransformation methodology, the DNA introduced into the cell contains agene that functions in a regenerable plant tissue to produce a compoundthat confers upon the plant tissue resistance to an otherwise toxiccompound. Genes of interest for use as a selectable, screenable, orscorable marker would include but are not limited to GUS, greenfluorescent protein (GFP), luciferase (LUX), antibiotic or herbicidetolerance genes. Examples of antibiotic resistance genes include thepenicillins, kanamycin (and neomycin, G418, bleomycin); methotrexate(and trimethoprim); chloramphenicol; kanamycin and tetracycline.Polynucleotide molecules encoding proteins involved in herbicidetolerance are known in the art, and include, but are not limited to apolynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) described in U.S. Pat. No. 5,627,061 (Barry, et al.),U.S. Pat. No. 5,633,435 (Barry, et al.), and U.S. Pat. No. 6,040,497(Spencer, et al.) and aroA described in U.S. Pat. No. 5,094,945 (Comai)for glyphosate tolerance; a polynucleotide molecule encoding bromoxynilnitrilase (Bxn) described in U.S. Pat. No. 4,810,648 (Duerrschnabel, etal.) for Bromoxynil tolerance; a polynucleotide molecule encodingphytoene desaturase (crtl) described in Misawa et al. (Plant J.4:833-840, 1993) and Misawa et al. (Plant Journal 6: 481-489, 1994) fornorflurazon tolerance; a polynucleotide molecule encodingacetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan etal. (Nucl. Acids Res. 18:2188-2193, 1990) for tolerance to sulfonylureaherbicides; and the bar gene described in DeBlock, et al. (EMBO J.6:2513-2519, 1987) for glufosinate and bialaphos tolerance.

The regeneration, development, and cultivation of plants from varioustransformed explants are well documented in the art. This regenerationand growth process typically includes the steps of selecting transformedcells and culturing those individualized cells through the usual stagesof embryonic development through the rooted plantlet stage. Transgenicembryos and seeds are similarly regenerated. The resulting transgenicrooted shoots are thereafter planted in an appropriate plant growthmedium such as soil. Cells that survive the exposure to the selectiveagent, or cells that have been scored positive in a screening assay, maybe cultured in media that supports regeneration of plants. Developingplantlets are transferred to soil less plant growth mix, and hardenedoff, prior to transfer to a greenhouse or growth chamber for maturation.

The present invention can be used with any transformable cell or tissue.By transformable as used herein is meant a cell or tissue that iscapable of further propagation to give rise to a plant. Those of skillin the art recognize that a number of plant cells or tissues aretransformable in which after insertion of exogenous DNA and appropriateculture conditions the plant cells or tissues can form into adifferentiated plant. Tissue suitable for these purposes can include butis not limited to immature embryos, scutellar tissue, suspension cellcultures, immature inflorescence, shoot meristem, nodal explants, callustissue, hypocotyl tissue, cotyledons, roots, and leaves.

Any suitable plant culture medium can be used. Examples of suitablemedia would include but are not limited to MS-based media (Murashige andSkoog, Physiol. Plant, 15:473-497, 1962) or N6-based media (Chu et al.,Scientia Sinica 18:659, 1975) supplemented with additional plant growthregulators including but not limited to auxins, cytokinins, ABA, andgibberellins. Those of skill in the art are familiar with the variety oftissue culture media, which when supplemented appropriately, supportplant tissue growth and development and are suitable for planttransformation and regeneration. These tissue culture media can eitherbe purchased as a commercial preparation, or custom prepared andmodified. Those of skill in the art are aware that media and mediasupplements such as nutrients and growth regulators for use intransformation and regeneration and other culture conditions such aslight intensity during incubation, pH, and incubation temperatures thatcan be optimized for the particular variety of interest.

One of ordinary skill will appreciate that, after an expression cassetteis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Identification of the Molecular Target Site for Picolinate Auxins.Identification of Picolinate Auxin Resistant Mutants

Mutations conferring picolinate auxin-specific resistance can beidentified by screening EMS-mutated M2 generation Arabidopsis seedlingsfor plants that can grow in the presence of a normally phytotoxic doseof a picolinate auxin. In a preferred embodiment, this can be a potentpicolinate auxin such as a 6-phenyl substituted auxin as described in WO03/011853 A1. Seedling survivors can be recovered and the progeny ofthese plants further tested for lack of resistance to 2,4-D. Thiseffectively eliminates previously discovered mutants such as axr1 thathave non-selective auxin-resistance. The chemical resistance spectrum ofmutants that are not resistant to 2,4-D can be characterized by testingwith a variety of different auxin herbicides. Genetic inheritance testscan determine if the mutations are dominant or recessive and the sitesof the mutations can be determined by genetic mapping.

The invention provides Arabidopsis nucleic acids encoding picolinateauxin resistance genes AFB5 and SGT1b, as presented in SEQ ID NO: 1(GenBank entry gi:30695799) and SEQ ID NO: 3 (GenBank entrygi:17017309). The invention further provides AFB5 and SGT1b proteinsequences, as presented in SEQ ID NO: 2 (gi:18423092) and SEQ ID NO: 4(gi:17017310), respectively. Nucleic acids and/or proteins that areorthologs or paralogs of Arabidopsis AFB5 and SGT1b have been identifiedand are described in Example 8 below. These orthologs or paralogsinclude AFB4 from Arabidopsis (SEQ ID NOs 5 and 6), homolog of AFB5 fromOryza sativa (rice) (GenBank entries gi|51979369 and gi134902411) (SEQID NOs 7, 8, 9 and 10) and an AFB5 homolog from Populus (GenBank entrygi|13249029 and gi|13249030) (SEQ ID NOs 11 and 12).

Across the entire length of the proteins, AFB4 and AFB5 share about 80%amino acid identity with each other and members of the AFB4 and AFB5group all have at least 54% identity with all other members of thegroup. For example, the protein sequence from Populus has about 70%identity with AFB4 and about 73% identity with AFB5. All members of thisgroup (SEQ ID NOs 5, 6, 7, 8, 9, 10, 11 and 12) are considered to bewithin, and usable according to, the subject invention.

As used herein, the term “AFB4, AFB5 and SGT1b polypeptides” refers to afull-length AFB4, AFB5 and SGT1b proteins or a fragments, derivatives(variants), or orthologs thereof that are “functionally active,” meaningthat the protein fragments, derivatives, or orthologs exhibit one ormore or the functional activities associated with the polypeptide of SEQID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. In one preferred embodiment, afunctionally active AFB4, AFB5 and SGT1b polypeptides cause an auxinresistant phenotype when mis-expressed in a plant. In a furtherpreferred embodiment, mis-expression of the AFB4, AFB5 or SGT1bpolypeptides causes an auxin-resistant phenotype in a plant. In anotherembodiment, a functionally active AFB4, AFB5 or SGT1b polypeptide iscapable of rescuing defective (including deficient) endogenous AFB4,AFB5 or SGT1b activity when expressed in a plant or in plant cells; therescuing polypeptide may be from the same or from a different species asthat with defective activity. In another embodiment, a functionallyactive fragment of a full length AFB4, AFB5 or SGT1b polypeptide (i.e.,a native polypeptide having the sequence of SEQ ID NO: 2, SEQ ID NO: 4or SEQ ID NO: 6 or naturally occurring orthologs thereof) retain one ofmore of the biological properties associated with the full-length AFB5or SGT1b polypeptides, such as signaling activity, binding activity,catalytic activity, or cellular or extra-cellular localizing activity.

Functionally active variants of full-length AFB4, AFB5 or SGT1bpolypeptides or fragments thereof include polypeptides with amino acidinsertions, deletions, or substitutions that retain one of more of thebiological properties associated with the full-length AFB4, AFB5 orSGT1b polypeptides. These polypeptides may be referred to as modifiedwild-type proteins, allelic variants or truncated polypeptides. In somecases, variants are generated that change the post-translationalprocessing of an AFB4, AFB5 or SGT1b polypeptides. For instance,variants may have altered protein transport or protein localizationcharacteristics or altered protein half-life compared to the nativepolypeptide.

Mutant forms of AFB4, AFB5 or SGT1b are referred to as AFB4-m, AFB5-mand SGT1b-m, respectively. The expression of AFB4-m, AFB5-m and SGT1b-min plants may cause resistance to picolinate auxins.

As used herein, the term “AFB4 nucleic acid”, “AFB5 nucleic acid” and“SGT1b nucleic acids encompass nucleic acids with the sequence providedin or complementary to the sequence provided in SEQ ID NO: 5, SEQ ID NO:1 and SEQ ID NO: 3, respectively, as well as functionally activefragments, derivatives, or orthologs thereof. An AFB4, AFB5 or SGT1bnucleic acid of this invention may be DNA, derived from genomic DNA orcDNA, or RNA.

In one embodiment, a functionally active AFB4, AFB5 or SGT1b nucleicacid encodes or is complementary to a nucleic acid that encodesfunctionally active AFB4, AFB5 or SGT1b polypeptides, respectively.Included within this definition is genomic DNA that serves as a templatefor a primary RNA transcript (i.e., an mRNA precursor) that requiresprocessing, such as splicing, before encoding the functionally activeAFB4, AFB5 or SGT1b polypeptide. An AFB4, AFB5 or SGT1b nucleic acid caninclude other non-coding sequences, which may or may not be transcribed;such sequences include 5′ and 3′ UTRs, polyadenylation signals andregulatory sequences that control gene expression, among others, as areknown in the art. Some polypeptides require processing events, such asproteolytic cleavage, covalent modification, etc., in order to becomefully active. Accordingly, functionally active nucleic acids may encodethe mature or the pre-processed AFB4, AFB5 or SGT1b polypeptide, or anintermediate form. An AFB4, AFB5 or SGT1b polynucleotide can alsoinclude heterologous coding sequences, for example, sequences thatencode a marker included to facilitate the purification of the fusedpolypeptide, or a transformation marker.

In another embodiment, a functionally active AFB4, AFB5 or SGT1b nucleicacid is capable of being used in the generation of loss-of-functionAFB4, AFB5 or SGT1b phenotypes, for instance, via antisense suppression,co-suppression, etc.

Identification of Orthologs that Bind Picolinate Auxins

Although AFB4, AFB5 and SGT1b genes were discovered in Arabidopsis andthe gene and protein information disclosed herein is information derivedfrom the Arabidopsis thaliana sequences, this invention is intended toencompass nucleic acid and protein sequences from other species ofplants that are similar to AFB4 (At4g24390; SEQ ID NO: 6), AFB5(At5g49980; SEQ ID NO: 2) and SGT1b (At4g11260; SEQ ID NO: 4). Someexamples are provided below in Example 8.

In one preferred embodiment, a AFB4, AFB5 or SGT1b nucleic acid used inthe methods of this invention comprises a nucleic acid sequence thatencodes or is complementary to a sequence that encodes AFB4, AFB5 orSGT1b polypeptides having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%,95% or more sequence identity to the polypeptide sequence presented inSEQ ID NO: 2 or SEQ ID NO: 4.

In another embodiment an AFB4, AFB5 or SGT1b polypeptide of theinvention comprises a polypeptide sequence with at least 50% or 60%identity to the AFB4, AFB5 or SGT1b polypeptide sequence of SEQ ID NO:6, SEQ ID NO: 2 or SEQ ID NO: 4, respectively, and may have at least70%, 80%, 85%, 90% or 95% or more sequence identity to the AFB4, AFB5 orSGT1b polypeptide sequence of SEQ ID NO: 6, SEQ ID NO: 2 or SEQ ID NO:4, respectively. In another embodiment, AFB4, AFB5 or SGT1b polypeptidescomprise a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%,90% or 95% or more sequence identity to a functionally active fragmentof the polypeptide presented in SEQ ID NO: 6, SEQ ID NO: 2 or SEQ ID NO:4, such as a F-box domain or a SGT1 domain, respectively. In yet anotherembodiment, an AFB4, AFB5 or SGT1b polypeptide comprises a polypeptidesequence with at least 50%, 60%, 70%, 80%, or 90% identity to thepolypeptide sequence of SEQ ID NO: 6, SEQ ID NO: 2 or SEQ ID NO: 4 overits entire length and comprises F box domain or SGT1 domain,respectively.

In another aspect, an AFB4, AFB5 or SGT1b polynucleotide sequence is atleast 50% to 60% identical over its entire length to the AFB4, AFB5 orSGT1b nucleic acid sequences presented as SEQ ID NO: 5, SEQ ID NO: 1 orSEQ ID NO: 3, respectively, or nucleic acid sequences that arecomplementary to such a AFB4, AFB5 or SGT1b sequences, and may compriseat least 70%, 80%, 85%, 90% or 95% or more sequence identity to theAFB4, AFB5 or SGT1b sequence presented as SEQ ID NO: 5, SEQ ID NO: 1 andSEQ ID NO: 3 or a functionally active fragment thereof, or complementarysequences.

As used herein, “percent (%) sequence identity” with respect to aspecified subject sequence, or a specified portion thereof, is definedas the percentage of nucleotides or amino acids in the candidatederivative sequence identical with the nucleotides or amino acids in thesubject sequence (or specified portion thereof), after aligning thesequences and introducing gaps, if necessary to achieve the maximumpercent sequence identity, as generated by the program WU-BLAST-2.0a19(Altschul et al., J. Mol. Biol. 215:403-410, 1997) with searchparameters set to default values. The HSP S and HSP S2 parameters aredynamic values and are established by the program itself depending uponthe composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched. A “% identity value” is determined by the number of matchingidentical nucleotides or amino acids divided by the sequence length forwhich the percent identity is being reported. “Percent (%) amino acidsequence similarity” is determined by doing the same calculation as fordetermining % amino acid sequence identity, but including conservativeamino acid substitutions in addition to identical amino acids in thecomputation. A conservative amino acid substitution is one in which anamino acid is substituted for another amino acid having similarproperties such that the folding or activity of the protein is notsignificantly affected. Aromatic amino acids that can be substituted foreach other are phenylalanine, tryptophan, and tyrosine; interchangeablehydrophobic amino acids are leucine, isoleucine, methionine, and valine;interchangeable polar amino acids are glutamine and asparagine;interchangeable basic amino acids are arginine, lysine and histidine;interchangeable acidic amino acids are aspartic acid and glutamic acid;and interchangeable small amino acids are alanine, serine, threonine,cysteine and glycine.

Derivative nucleic acid molecules of the subject nucleic acid moleculesinclude sequences that hybridize to the nucleic acid sequence of SEQ IDNO: 5, SEQ ID NO: 1 or SEQ ID NO: 3. The stringency of hybridization canbe controlled by temperature, ionic strength, pH, and the presence ofdenaturing agents such as formamide during hybridization and washing.Conditions routinely used are well known (see, e.g., Current Protocol inMolecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers,1994; Sambrook et al., Molecular Cloning, Cold Spring Harbor, 1989). Insome embodiments, a nucleic acid molecule of the invention is capable ofhybridizing to a nucleic acid molecule containing the nucleotidesequence of SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3 under stringenthybridization conditions that are: prehybridization of filterscontaining nucleic acid for 8 hours to overnight at 65° C. in a solutioncomprising 6× single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015M Na citrate; pH 7.0), 5×Denhardt's solution, 0.05% sodium pyrophosphateand 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C.in a solution containing 6×SSC, 1×Denhardt's solution, 100 μg/ml yeasttRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C.for 1 h in a solution containing 0.1×SSC and 0.1% SDS (sodium dodecylsulfate). In other embodiments, moderately stringent hybridizationconditions are used that are: pretreatment of filters containing nucleicacid for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. ina solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA,and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hourat 55° C. in a solution containing 2×SSC and 0.1% SDS. Alternatively,low stringency conditions can be used that comprise: incubation for 8hours to overnight at 37° C. in a solution comprising 20% formamide,5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA;hybridization in the same buffer for 18 to 20 hours; and washing offilters in 1×SSC at about 37° C. for 1 hour.

As a result of the degeneracy of the genetic code, a number ofpolynucleotide sequences encoding an AFB4, AFB5 or SGT1b polypeptidescan be produced. For example, codons may be selected to increase therate at which expression of the polypeptide occurs in a particular hostspecies, in accordance with the optimum codon usage dictated by theparticular host organism (see, e.g., Nakamura et al. N A R 27: 292,1999). Such sequence variants may be used in the methods of thisinvention.

The methods of the invention may use orthologs of the Arabidopsis AFB4,AFB5 or SGT1b. Methods of identifying the orthologs in other plantspecies are known in the art. Normally, orthologs in different speciesretain the same function, due to presence of one or more protein motifsand/or 3-dimensional structures. In evolution, when a gene duplicationevent follows speculation, a single gene in one species, such asArabidopsis, may correspond to multiple genes (paralogs) in another. Asused herein, the term “orthologs” encompasses paralogs. When sequencedata is available for a particular plant species, orthologs aregenerally identified by sequence homology analysis, such as BLASTanalysis, usually using protein bait sequences. Sequences are assignedas a potential ortholog if the best hit sequence from the forward BLASTresult retrieves the original query sequence in the reverse BLAST(Huynen M A and Bork P, Proc Natl Acad Sci, 95:5849-5856, 1998; Huynen MA et al., Genome Research, 10:1204-1210, 2000). Programs for multiplesequence alignment, such as CLUSTAL (Thompson J D et al, Nucleic AcidsRes 22:4673-4680, 1994) may be used to highlight conserved regionsand/or residues of orthologous proteins and to generate phylogenetictrees. In a phylogenetic tree representing multiple homologous sequencesfrom diverse species (e.g., retrieved through BLAST analysis),orthologous sequences from two species generally appear closest on thetree with respect to all other sequences from these two species.Structural threading or other analysis of protein folding (e.g., usingsoftware by ProCeryon, Biosciences, Salzburg, Austria) may also identifypotential orthologs. Nucleic acid hybridization methods may also be usedto find orthologous genes and are preferred when sequence data are notavailable. Degenerate PCR and screening of cDNA or genomic DNA librariesare common methods for finding related gene sequences and are well knownin the art (see, e.g., Sambrook et al., Molecular Cloning, Cold SpringHarbor, 1989; Dieffenbach and Dveksler, PCR Methods and Applications 3:S2-S7, 1993). For instance, methods for generating a cDNA library fromthe plant species of interest and probing the library with partiallyhomologous gene probes are described in Sambrook et al., (MolecularCloning, Cold Spring Harbor, 1989). A highly conserved portion of theArabidopsis AFB4, AFB5 or SGT1b coding sequences may be used as probes.AFB4, AFB5 or SGT1b ortholog nucleic acids may hybridize to the nucleicacid of SEQ ID NO: 5, SEQ ID NO: 1 or SEQ ID NO: 3 under high, moderate,or low stringency conditions. After amplification or isolation of asegment of a putative ortholog, that segment may be cloned and sequencedby standard techniques and utilized as a probe to isolate a completecDNA or genomic clone. Alternatively, it is possible to initiate an ESTproject to generate a database of sequence information for the plantspecies of interest. In another approach, antibodies that specificallybind known AFB4, AFB5 or SGT1b polypeptides are used for orthologisolation (see, e.g., Harlow & Lane, Cold Spring Harbor Laboratory, NY,1988, 1999). Western blot analysis can determine that AFB4, AFB5 orSGT1b orthologs (i.e., an orthologous protein) is present in a crudeextract of a particular plant species. When reactivity is observed, thesequence encoding the candidate ortholog may be isolated by screeningexpression libraries representing the particular plant species.Expression libraries can be constructed in a variety of commerciallyavailable vectors, including lambda gt11, as described in Sambrook, etal., (Molecular Cloning, Cold Spring Harbor, 1989). Once the candidateortholog(s) are identified by any of these means, candidate orthologoussequence are used as bait (the “query”) for the reverse BLAST againstsequences from Arabidopsis or other species in which AFB4, AFB5 or SGT1bnucleic acids and/or polypeptide sequences have been identified.

AFB4, AFB5 or SGT1b nucleic acids and polypeptides may be obtained usingany available method. For instance, techniques for isolating cDNA orgenomic DNA sequences of interest by screening DNA libraries or by usingpolymerase chain reaction (PCR), as previously described, are well knownin the art. Alternatively, nucleic acid sequence may be synthesized. Anyknown method, such as site directed mutagenesis (Kunkel et al., Methodsin Enzymology 204: 125-139, 1991), may be used to introduce desiredchanges into a cloned nucleic acid.

In general, the methods of the invention involve incorporating thedesired form of the AFB4, AFB5 or SGT1b nucleic acids into a plantexpression vector for transformation of in plant cells, and the AFB4,AFB5 or SGT1b polypeptides are expressed in the host plant.

An “isolated” AFB4, AFB5 or SGT1b nucleic acid molecule or protein isother than in the form or setting in which it is found in nature. An“isolated” polynucleotide is and separated from least one contaminantnucleic acid molecule with which it is ordinarily associated in thenatural source of the AFB4, AFB5 or SGT1b nucleic acid. However, anisolated AFB4, AFB5 or SGT1b nucleic acid molecule includes AFB4, AFB5or SGT1b nucleic acid molecules contained in cells that ordinarilyexpress AFB4, AFB5 or SGT1b where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

Chemical Structures of Picolinate Auxins, and Identification of theBinding Site for Picolinate Auxins on AFB4 or AFB5

The binding site of picolinate auxins on AFB4 or AFB5 may be identifiedby dissecting functional domains of the protein. Assays described inDharmasiri et al. (2005 Nature 435:441-445) and Kepinski and Leyser(Nature, 435:446-451, 2005) have demonstrated that the natural auxin IAAbinds directly to TIR1. Similar assays will be used to demonstrate thatpicolinate auxins bind to AFB4 or AFB5. Briefly, fusion proteins of AFB4or AFB5 and the myc epitope will be made and tested for ability tointeract with an IAA protein in a picolinate auxin-dependent manner. Theinteraction of AFB4 or AFB5 and AUX/IAA proteins may be tested in vitroby “pull down” experiments. Arabidopsis has approximately 25 AUX/IAAproteins and AFB5 may interact with some or all of them. AUX2 and AUX3are commonly used in “pull down” experiments with TIR1 and can be usedin experiments with AFB4 or AFB5. In “pull down” experiments, antibodiesto one protein are used to immunoprecipitate a protein complex. Theprecipitated proteins are separated by electrophoresis and otherproteins in the complex are detected by immunoblot analysis. The “pulldown” experiments may be performed with the cloned proteins andantiserum specific for them used for immunoprecipitation andimmunodetection. Alternatively epitopes recognized by commerciallyavailable antiserum may be fused to the proteins and a commerciallyavailable antisera used. For example, sequences encoding the myc epitope(EQKLISEEDL) (SEQ ID NO: 13) may be fused with sequences encoding AFB4or AFB5 such that the myc epitope is fused at the N or preferably at theC terminus of the AFB4 or AFB5 protein forming AFB4-myc or AFB5-mycfusion proteins, respectively. Similarly, sequences encoding theglutathione S transferase (GST) protein may be fused to sequencesencoding the AUX2 protein such that the GST protein is fused at the Cterminus of the AUX2 protein. The AFB4-myc or AFB5-myc fusion protein ismade by transcribing and translating the gene in a non-plant system.Transcripts encoding the AFB4-myc or AFB5-myc fusion protein may betranslated in a cell free system, such as the wheat germ or reticulatelysate systems or the AFB5-myc gene may be transformed into E. coli,yeast, insect cells or some other appropriate host and the fusionprotein purified from cell extracts. The AUX2-GST fusion protein may bepurified similarly. The interaction of the AFB4-myc or AFB5-myc proteinwith AUX2-GST fusion may be tested by incubating the two proteins in thepresence and absence of picolinate auxins such as DAS534,immunoprecipitating protein complexes with the commercially availablemyc antiserum, resuspending the immunoprecipitate and separating theproteins by electrophoresis. Immunoblot analysis using anti-GSTantiserum will determine how much of the AUX2-GST fusion protein isinteracting with the AFB4-myc or AFB5-myc protein. Detecting moreAUX2-GST protein when DAS534 is included in the assay will indicate thatDAS534 stimulates the interaction of AFB4 or AFB5 and AUX2. As acontrol, similar experiments can be done with TIR1-myc fusion proteins.The TIR1/AUX2 interaction is stimulated weakly or not at all bypicolinate auxins.

To determine which portion of the AFB4 or AFB5 protein interacts withpicolinate auxins, fusion proteins composed of portions of AFB5 and TIR1can be constructed. For example the N-terminal portion of TIR1 encodingthe F-box can be attached to the C terminal portion of AFB5 encoding theleucine rich repeat (LRR) domain. If the fusion protein should interactless strongly with AUX2 in the presence of picolinate auxins, than thewild-type protein, it may indicate that picolinate auxins interact withthe AFB4 or AFB5 in the F-box portion of the protein. Similarly, theN-terminal, F-box encoding portion of AFB4 or AFB5 is fused with theC-terminal, LRR domain encoding portion of TIR1 and be used in “pulldown” experiments. In this case, if the fusion protein interacts withAUX2 more strongly than the wild-type TIR1 protein, it will indicatethat picolinate auxins interact with F-box proteins through the F-boxdomain. Using chimeric AFB4-TIR1 or AFB5-TIR1 fusion proteins similar tothe ones described above, the picolinate auxin binding site on AFB5 maybe determined.

The herbicide DAS534 (compound 173 in PCT publication WO 2003011853),4-amino-3-chloro-5-fluoro-6-(4-chlorophenyl)pyridine-2-carboxylic acid,has the following chemical structure:

“Picolinate auxins” contain the core structure, 3-chloro-2-picolinicacid:

“Aryloxyalkanoate auxins” contain the core structures,4-chlorophenoxyacetic acid or 2-(4-chlorophenoxy)-propionic acid or5-chloro-2-pyridinyloxyacetic acid:

Functional Assay of AFB5 to Identify Other Herbicidal Compounds

The “pull down” experiments described above can be used to identifyother compounds that stimulate interaction between AFB5 and AUX/IAAproteins. In these experiments, test compounds would be added to themixture of AFB4 or AFB5 and AUX2. Antiserum to the AFB4 or AFB5 proteinwould be used to immunoprecipitate protein complexes. These complexeswould be resuspended and the proteins separated by electrophoresis.Immunodetection using antiserum to the AUX2 protein would be used toquantitate the amount of AUX2 protein interacting with AFB4 or AFB5.Compounds that stimulate the interaction would be evaluated for auxinicactivity on whole plants. Although any compounds could be screened inthis assay, the preferred embodiment would be compounds that contain apicolinate or similar moiety.

Ligand-Binding Assay of AFB5 to Identify Herbicidal Compounds

Binding of compounds to AFB4 or AFB5 can be detected by liganddisplacement techniques wherein a picolinate auxin is labeled with aradioisotope such as tritium or with a tag such as biotin or with afluorescent moiety. The amount of binding of the picolinate auxin toAFB4 or AFB5 can be detected by separation of the protein from themedium by immunoprecipitation or other protein capture methods.Alternatively alterations in a fluorescent signal from afluorescence-tagged picolinate on binding to AFB4 or AFB5 can provide asignal for ligand-binding. These detection methods for ligand-bindingprovide convenient and high-throughput techniques for screeningcompounds to identify those that inhibit binding of the taggedpicolinate auxin to AFB4 or AFB5. These experiments may be facilitatedby heterologous expression of AFB4 or AFB5 proteins in a suitable hostsystem such as E. coli or Saccharomyces cerevisiae or via baculovirusexpression in insect cell cultures.

Combining In Vitro Assays to Identify Potent Broad Spectrum Auxins

There is redundancy in auxin receptors (AFBs) at the gene and proteinlevel. Current herbicidal auxins interact only with a subset of theseproteins for example 2,4-D interacts with TIR1 and close homologswhereas picloram preferentially interacts with AFB4 or AFB5.Consequently a preferred embodiment is to select compounds foroptimization by identifying those that interact with TIR1, AFB4 and AFB5in biochemical ligand-binding or functional assays. This will identifycompounds such as DAS534 that interact strongly with both classes ofreceptor and thus have the potential for increased potency and/orbroader spectrum.

Co-Crystals of Picolinates and AFB4 or AFB5

Purification of AFB4 or AFB5, especially from heterologous expressionsystems, allows the protein to be crystallized either on its own orwithin the E3 protein ligase complex with which it associates. See forexample the crystallization of the SCF complex described in Zheng et al.(Nature 416:703-708, 2002). The protein structure can then be determinedby X-ray crystallography. Crystallization can be done in the presenceand absence of a picolinate auxin ligand by co-crystallography or byligand-soaking into preformed crystals. This enables the binding regioninvolved in the protein-ligand interaction to be discerned. The featuresof this molecular interaction can then be used for the design andsynthesis of novel compounds that can occupy the site to effectherbicidal activity. Alternatively, the binding pocket can be used as atemplate to computationally screen and identify compounds with thepotential to bind to the site. These can then be experimentally verifiedas binding to the protein by further in vitro or in vivo assays.

Generation of Genetically Modified Plants with a PicolinateAuxin-Resistant Phenotype

AFB4, AFB5 or SGT1b nucleic acids and polypeptides may be used in thegeneration of genetically modified plants having an auxin-resistantphenotype. As used herein, an “auxin resistant phenotype” may refer toresistance to any naturally occurring or synthetic molecule that elicitsauxinic herbicidal symptoms when applied to plants. A preferredembodiment is for resistance to compounds containing a picolinate orsimilar moiety.

The methods described herein are generally applicable to all plants.Although EMS mutagenesis and gene identification was carried out inArabidopsis, the AFB4, AFB5 and SGT1b genes (or an ortholog, variant orfragment thereof) may be expressed in any type of plant. In a preferredembodiment, the invention is directed to plants known as row crops.Examples of row crop species include soybean (Glycine max), rapeseed andcanola (including Brassica napus, B. campestris), sunflower (Helianthusannuus), cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobromacacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis),coconut palm (Cocos nucifera), flax (Linum usitatissimum), castor(Ricinus communis), sorghum (Sorghum bicolor) and peanut (Arachishypogaea). The invention may also be directed to fruit- andvegetable-bearing plants, grain-producing plants, nut-producing plants,rapid cycling Brassica species, alfalfa (Medicago sativa), tobacco(Nicotiana spp.), turfgrass (Poaceae family), clover (Trifolium spp.)and other forage crops.

The auxin-resistant phenotype may be generated by expressing AFB4, AFB5or SGT1b gene in antisense or hairpin RNAs, by over-expressing AFB4,AFB5 or SGT1b and causing co-suppression, or by developing a dominantnegative protein that inhibits the function of the native protein.Antisense RNAs are formed when the coding sequences of a proteinencoding nucleic acid are expressed in the opposite orientation. TheseRNAs can hybridize with mRNAs transcribed from endogenous genes andinhibit their expression. Hairpin RNAs are formed when all or a portionof the coding sequences of a gene are cloned in the sense and antisenseorientation in the same transcription unit. Upon transcription of theseconstructs, the transcript forms a hairpin structure and triggers atranscript degradation pathway that targets both the hairpin-encodinggene and the endogenous gene. This degradation pathway can effectivelysilence endogenous gene expression (Smith et al. Nature 407:319-320,2000). Co-suppression is caused by the over-expression of a transgene;high level of expression can cause the degradation of transcripts fromthe native gene (Que et al. Plant Cell 9:1357-1368, 1997). Dominantnegative mutants may be generated by over-expressing a mutant form ofthe protein (Pontier et al., Plant Cell 27:529-538, 2001). Since AFB4,AFB5 and SGT1b interact with components of the SCF complex,over-expression of mutant forms of the proteins may disrupt theauxin-responsive SCF complex, prevent auxin signaling and cause thetransgenic plants to be auxin resistant.

The skilled artisan will recognize that a wide variety of transformationtechniques exist in the art, and new techniques are continually becomingavailable. Any technique that is suitable for the target host plant canbe employed within the scope of the present invention. For example, theconstructs can be introduced in a variety of forms including, but notlimited to as a strand of DNA, in a plasmid, or in an artificialchromosome. The introduction of the constructs into the target plantcells can be accomplished by a variety of techniques, including, but notlimited to Agrobacterium-mediated transformation, electroporation,microinjection, microprojectile bombardment calcium-phosphate-DNAco-precipitation or liposome-mediated transformation of a heterologousnucleic acid. The transformation of the plant is preferably permanent,i.e. by integration of the introduced expression constructs into thehost plant genome, so that the introduced constructs are passed ontosuccessive plant generations. Depending upon the intended use, aheterologous nucleic acid construct comprising an AFB4, AFB5 or SGT1bpolynucleotide may encode the entire protein or a biologically activeportion thereof.

In one embodiment, binary Ti-based vector systems may be used totransfer polynucleotides. Standard Agrobacterium binary vectors areknown to those of skill in the art, and many are commercially available(e.g., pBI121 Clontech Laboratories, Palo Alto, Calif.). A construct orvector may include a plant promoter to express the nucleic acid moleculeof choice. In a preferred embodiment, the promoter is a plant promoter.

The optimal procedure for transformation of plants with Agrobacteriumvectors will vary with the type of plant being transformed. Exemplarymethods for Agrobacterium-mediated transformation include transformationof explants of hypocotyl, shoot tip, stem or leaf tissue, derived fromsterile seedlings and/or plantlets. Such transformed plants may bereproduced sexually, or by cell or tissue culture. Agrobacteriumtransformation has been previously described for a large number ofdifferent types of plants and methods for such transformation may befound in the scientific literature. Of particular relevance are methodsto transform commercially important crops, such as rapeseed (De Block etal., Plant Physiol 91: 694-701, 1989), sunflower (Everett et al.,Bio/Technology 5: 1201-1204, 1987), and soybean (Christou et al., ProcNatl Acad Sci USA 86: 7500-7504, 1989; Kline et al., Nature 327: 70-73,1987).

Expression (including transcription and translation) of AFB4, AFB5 orSGT1b may be regulated with respect to the level of expression, thetissue type(s) where expression takes place and/or developmental stageof expression. A number of heterologous regulatory sequences (e.g.,promoters and enhancers) are available for controlling the expression ofan AFB4, AFB5 or SGT1b nucleic acid. These include constitutive,inducible and regulatable promoters, as well as promoters and enhancersthat control expression in a tissue- or temporal-specific manner.Exemplary constitutive promoters include, but are not limited to, theraspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and 5,783,394), thenopaline synthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci.(U.S.A.) 84:5745-5749, 1987), the octopine synthase (OCS) promoter(which is carried on tumor-inducing plasmids of Agrobacteriumtumefaciens), the caulimovirus promoters such as the cauliflower mosaicvirus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324,1987) and the CaMV ³⁵S promoter (Odell et al., Nature 313:810-812, 1985and Jones et al, Transgenic Res. 1(6):285-297, 1992), the melon actinpromoter (published PCT application WO 00/56863), the figwort mosaicvirus 35S-promoter (U.S. Pat. No. 5,378,619), the light-induciblepromoter from the small subunit of ribulose-1,5-bis-phosphatecarboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl.Acad. Sci. (U.S.A.) 84:6624-6628, 1987), the sucrose synthase promoter(Yang et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:4144-4148, 1990), the Rgene complex promoter (Chandler et al., The Plant Cell 1:1175-1183,1989), the chlorophyll a/b binding protein gene promoter the CsVMVpromoter (Verdaguer et al., Plant Mol Biol 37: 1055-1067, 1998), thesepromoters have been used to create DNA constructs that have beenexpressed in plants; see, e.g., PCT publication WO 84/02913. Exemplarytissue-specific promoters include the tomato E4 and E8 promoters (U.S.Pat. No. 5,859,330) and the tomato 2AII gene promoter (Van Haaren etal., Plant Mol Biol 21: 625-640, 1993).

In yet another aspect, in some cases it may be desirable to inhibit theexpression of endogenous AFB4, AFB5 or SGT1b in a host cell. Exemplarymethods for practicing this aspect of the invention include, but are notlimited to antisense suppression (Smith, et al., Mol Gen Genet. 224:477-481, 1988; van der Krol et al., BioTechniques 6: 958-976, 1988);co-suppression (Napoli, et al., Plant Cell 2: 279-289, 1990); ribozymes(PCT Publication WO 97/10328); and combinations of sense and antisense(Waterhouse, et al., Proc Natl Acad Sci USA 95: 13959-13964, 1998).Methods for the suppression of endogenous sequences in a host celltypically employ the transcription or transcription and translation ofat least a portion of the sequence to be suppressed. Such sequences maybe homologous to coding as well as non-coding regions of the endogenoussequence. Antisense inhibition may use the entire cDNA sequence (Sheehyet al., Proc Natl Acad Sci USA 85: 8805-8809, 1988), a partial cDNAsequence including fragments of 5′ coding sequence, (Cannon et al.,Plant Mol Biol 15: 39-47, 1990), or 3′ non-coding sequences (Ch'ng etal., Proc Natl Acad Sci USA 86: 10006-10010, 1989). Cosuppressiontechniques may use the entire cDNA sequence (Napoli et al., Plant Cell2: 279-289, 1990; van der Krol et al., Plant Cell 2: 291-299, 1990), ora partial cDNA sequence (Smith et al., Mol Gen Genet. 224: 477-481,1990).

AFB4, AFB5 or SGT1b activity can be decreased by decreasing in theexpression levels of AFB4, AFB5 or SGT1b and/or decreasing the activityof AFB4, AFB5, or SGT1b collectively these are referred to as “genedown-regulation mechanisms”. Decreased activity of AFB4, AFB5 or SGT1bcan be obtained, for example, by decreasing expression of the AFB4, AFB5or SGT1b polypeptides, or variants or fragments thereof. For example, anucleotide sequence may be introduced into a cell or plant that encodesa protein that leads to decreased AFB4, AFB5, or SGT1b expression, suchas a protein that interferes with the AFB4, AFB5 or SGT1b promoter.Alternatively, a non-protein-encoding nucleotide sequence that decreases(inhibits) expression of AFB4, AFB5 or SGT1b, or variant or fragmentthereof, may be introduced into a cell or plant. The inhibitorynucleotide sequence may hybridize to a nucleotide sequence encoding theAFB4, AFB5 or SGT1b polypeptide, or variant or fragment thereof.Inhibitory nucleotide sequences include, but are not limited to, anantisense nucleotide sequence, a siRNA or a ribozyme. A chemicalcompound that is capable of decreasing AFB4, AFB5 or SGT1b expressionmay be introduced into the cell or plant.

In one embodiment, inhibition of AFB4, AFB5, or SGT1b activity isobtained through the use of antisense technology. Antisenseoligonucleotides as a method of suppression are well known in the art.Although the exact mechanism by which antisense RNA molecules interferewith gene expression has not been elucidated, it is believed thatantisense RNA molecules bind to the endogenous mRNA molecules andthereby inhibit translation of the endogenous mRNA.

Herein is provided a means to alter levels of expression of AFB4, AFB5,or SGT1b by the use of a synthetic antisense oligonucleotide compoundthat inhibits translation of mRNA encoding AFB4, AFB5, or SGT1b.Synthetic antisense oligonucleotides, or other antisense chemicalstructures designed to recognize and selectively bind to mRNA, areconstructed to be complementary to portions of the nucleotide sequenceshown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. In addition, theantisense oligonucleotide may be designed for administration only tocertain selected cell populations by targeting the antisenseoligonucleotide to be recognized by specific cellular uptake mechanismsthat bind and take up the antisense oligonucleotide only within certainselected cell populations. For example, the antisense oligonucleotidemay be designed to bind to transporter found only in a certain celltype. The antisense oligonucleotide may be designed to inactivate theAFB4, AFB5, or SGT1b mRNA by (1) binding to the AFB4, AFB5, or SGT1bmRNA and thus inducing degradation of the mRNA by intrinsic cellularmechanisms such as RNase I digestion, (2) by inhibiting translation ofthe mRNA target by interfering with the binding oftranslation-regulating factors or of ribosomes, or (3) by inclusion ofother chemical structures, such as ribozyme sequences or reactivechemical groups, which either degrade or chemically modify the targetmRNA. Synthetic antisense oligonucleotide drugs have been shown to becapable of the properties described above when directed against mRNAtargets (Cohen, Trends Pharmacol. Sci. 10:435-437, 1989). In addition,coupling of ribozymes to antisense oligonucleotides is a promisingstrategy for inactivating target mRNA (Sarver et al., Science247:1222-1225, 1990). In this manner, an antisense oligonucleotidedirected to AFB4, AFB5, or SGT1b can be used to reduce AFB4, AFB5, orSGT1b expression in particular target cells.

The introduced sequence need not be the full-length AFB4, AFB5, or SGT1bcDNA (SEQ ID NO: 5, SEQ ID NO: 1 and SEQ ID NO: 3, respectively) andneed not be exactly homologous to the equivalent sequence found in thecell type to be transformed. The sequence may comprise a flanking regionof AFB4, AFB5, or SGT1b. Flanking regions of AFB4, AFB5, or SGT1b in anytarget plant may be obtained using the sequences provided herein.

In one embodiment, portions or fragments of the AFB4, AFB5, or SGT1bcDNA (SEQ ID NO: 5, SEQ ID NO: 1 and SEQ ID NO: 3, respectively) couldbe used to knock out expression of the AFB4, AFB5, or SGT1b gene.Generally, however, where the introduced sequence is of shorter length,a higher degree of identity to the native AFB4, AFB5, or SGT1b sequencewill be needed for effective antisense suppression. In otherembodiments, the introduced antisense sequence in the vector may be atleast 15 nucleotides in length, and improved antisense suppressiontypically will be observed as the length of the antisense sequenceincreases. In yet other embodiments, the length of the antisensesequence in the vector advantageously may be greater than 100nucleotides, and can be up to about the full length of the AFB4, AFB5,or SGT1b cDNA or gene. In another embodiment, for suppression of theAFB4, AFB5, or SGT1b gene itself, transcription of an antisenseconstruct results in the production of RNA molecules that are thereverse complement of mRNA molecules transcribed from the endogenousAFB4, AFB5, or SGT1b gene in the cell.

In one embodiment, the oligonucleotide is at least about 10 nucleotidesin length, such as, greater than about 20 bases in length, greater thanabout 30 bases in length, greater than about 40 bases in length, greaterthan about 50 bases in length, greater than about 100 bases in length,greater than about 200 bases in length or greater than about 300 basesin length. In one embodiment, the oligonucleotide has a ribozymeactivity.

In one specific, non-limiting embodiment, a nucleotide sequence from anAFB4, AFB5, or SGT1b encoding sequence is arranged in reverseorientation relative to the promoter sequence in the transformationvector.

The synthesis of effective anti-sense inhibitors is known. Syntheticantisense oligonucleotides may be produced, for example, in acommercially available oligonucleotide synthesizer. Numerous approacheshave been previously described and generally involve altering thebackbone of the polynucleotide to increase its stability in vivo.Exemplary oligonucleotides and methods of synthesis are described inU.S. Pat. Nos. 5,661,134; 5,635,488; and 5,599,797 (phosphorothioatelinkages), U.S. Pat. Nos. 5,587,469 and 5,459,255 (N-2 substitutedpurines), U.S. Pat. No. 5,539,083 (peptide nucleic acids) and U.S. Pat.Nos. 5,629,152; 5,623,070; and 5,610,289 (miscellaneous approaches).

In another embodiment, suppression of endogenous AFB4, AFB5, or SGT1bexpression can be achieved using ribozymes. Ribozymes are synthetic RNAmolecules that possess highly specific endoribonuclease activity. Theproduction and use of ribozymes are disclosed in U.S. Pat. No. 4,987,071to Cech and U.S. Pat. No. 5,543,508 to Haselhoff. In one embodiment, theinclusion of ribozyme sequences within antisense RNAs may be used toconfer RNA cleaving activity on the antisense RNA, such that endogenousmRNA molecules that bind to the antisense RNA are cleaved, which in turnleads to an enhanced antisense inhibition of endogenous gene expression.In yet another embodiment, dominant negative mutant forms of AFB4, AFB5,or SGT1b may be used to block endogenous AFB4, AFB5, or SGT1b activity.

Decreased activity of AFB4, AFB5, or SGT1b may also be obtained bydecreasing the activity of the AFB4, AFB5, or SGT1b protein with orwithout decreasing expression levels. For example, a nucleotide sequenceencoding a protein that inhibits or inactivates AFB4, AFB5, or SGT1bactivity may be introduced into the cell or plant.

In one embodiment, a mutant or variant of AFB4, AFB5, or SGT1b isintroduced or expressed in a cell. The variant of AFB4, AFB5, or SGT1boptionally may have increased expression or decreased expression. Cellsmay have AFB4, AFB5, or SGT1b null mutations, AFB4, AFB5, or SGT1bmissense mutations, AFB4, AFB5, or SGT1b truncation mutations, orinactivation of AFB4, AFB5, or SGT1b. In one embodiment, a mutant AFB4,AFB5, or SGT1b is expressed in a cell but is incapable of localizing tothe correct subcellular location. In another embodiment, a mutant AFB4,AFB5, or SGT1b is incapable of binding to one or more of itsintracellular binding partners. In yet another embodiment, a mutation inthe upstream regulatory region of the AFB4, AFB5, or SGT1b geneabrogates the expression of the protein. In another embodiment, a mutantAFB4, AFB5, or SGT1b is incapable of forming an active SCF complex.

It is possible that mutations in the AFB4, AFB5, or SGT1b gene are notincluded in the cDNA but rather are located in other regions of theAFB4, AFB5, or SGT1b gene. Mutations located outside of the ORF thatencode the AFB4, AFB5, or SGT1b protein are not likely to affect thefunctional activity of the proteins, but rather are likely to result inaltered levels of the proteins in the cell. For example, mutations inthe promoter region of the AFB4, AFB5, or SGT1b gene may preventtranscription of the gene and therefore lead to the complete absence ofthe AFB4, AFB5, or SGT1b protein, or absence of certain transcripts ofthe protein, in the cell.

Additionally, mutations within introns in the genomic sequence may alsoprevent expression of the AFB4, AFB5, or SGT1b protein. Followingtranscription of a gene containing introns, the intron sequences areremoved from the RNA molecule, in a process termed splicing, prior totranslation of the RNA molecule that results in production of theencoded protein. When the RNA molecule is spliced to remove the introns,the cellular enzymes that perform the splicing function recognizesequences around the intron/exon border and in this manner recognize theappropriate splice sites. If a mutation exists within the sequence ofthe intron near and exon/intron junction, the enzymes may not recognizethe junction and may fail to remove the intron. If this occurs, theencoded protein will likely be defective. Thus, mutations inside theintron sequences within the AFB4, AFB5, or SGT1b gene (termed “splicesite mutations”) may also lead to defects in transporter activity.However, knowledge of the exon structure and intronic splice sitesequences of the AFB4, AFB5, or SGT1b gene is required to define themolecular basis of these abnormalities. The provision herein of AFB4,AFB5, or SGT1b cDNA sequences enables the cloning of the entire AFB4,AFB5, or SGT1b gene (including the promoter and other regulatory regionsand the intron sequences) from any target plant, and the determinationof its nucleotide sequence.

Standard molecular and genetic tests may be performed to further analyzethe association between a gene and an observed phenotype. Exemplarytechniques are described below.

1. DNA/RNA Analysis

The stage- and tissue-specific gene expression patterns in mutant versuswild-type lines may be determined, for instance, by in situhybridization. Analysis of the methylation status of the gene,especially flanking regulatory regions, may be performed. Other suitabletechniques include overexpression, ectopic expression, expression inother plant species and gene knock-out (reverse genetics, targetedknock-out, viral induced gene silencing [VIGS, see Baulcombe D, CurrentOpinion in Plant Biology 2: 109-113, 1999]).

In one preferred application, expression profiling (generally bymicroarray analysis) is used to simultaneously measure differences orinduced changes in the expression of many different genes. Techniquesfor microarray analysis are well known in the art (Schena et al.,Science 270:467-470, 1995; Baldwin et al., Curr Opin Plant Biol.2(2):96-103, 1999; Dangond, Physiol Genomics 2:53-58, 2000; van Hal etal., J Biotechnol 78:271-280, 2000; Richmond & Somerville, Curr OpinPlant Biol 3:108-116, 2000). Expression profiling of individual taggedlines may be performed. Such analysis can identify other genes that arecoordinately regulated as a consequence of the over-expression of thegene of interest, which may help to place an unknown gene in aparticular pathway.

2. Gene Product Analysis

Analysis of gene products may include recombinant protein expression,antisera production, immunolocalization, biochemical assays forcatalytic or other activity, analysis of phosphorylation status, andanalysis of interaction with other proteins via yeast two-hybrid assays.

3. Pathway Analysis

Pathway analysis may include placing a gene or gene product within aparticular biochemical, metabolic or signaling pathway based on itsmis-expression phenotype or by sequence homology with related genes.Alternatively, analysis may comprise genetic crosses with wild-typelines and other mutant lines (creating double mutants) to order the genein a pathway, or determining the effect of a mutation on expression ofdownstream “reporter” genes in a pathway.

Generation of Mutated Plants with a Picolinate-Resistance Phenotype

The invention further provides a method of identifying plants that havemutations in endogenous AFB4, AFB5 or SGT1b that confer auxinresistance. In one method, called “TILLING” (for Targeting Induced LocalLesions IN Genomes), mutations are induced in the seed of a plant ofinterest, for example, using EMS treatment. The resulting plants aregrown and self-fertilized, and the progeny are used to prepare DNAsamples. Gene specific PCR is used to identify whether a mutated planthas an AFB4, AFB5 or SGT1b mutation. Plants having AFB4, AFB5 or SGT1bmutations may then be tested for auxin resistance, or alternatively,plants may be tested for auxin resistance, and then AFB4, AFB5 orSGT1b-specific PCR is used to determine whether a resistant plant has amutated AFB4, AFB5 or SGT1b gene. TILLING can identify mutations thatmay alter the expression of specific genes or the activity of proteinsencoded by these genes (see Colbert et al., Plant Physiol 126:480-484,2001; McCallum et al., Nature Biotechnology 18:455-457, 2000).

In another method, a candidate gene/Quantitative Trait Locus (QTLs)approach can be used in a marker-assisted breeding program to identifyalleles of or mutations in AFB4, AFB5 or SGT1b or orthologs that mayconfer auxin resistance (see Bert et al., Theor Appl Genet.,107(1):181-9, 2003; and Lionneton et al., Genome. 45(6):1203-15, 2002).Thus, in a further aspect of the invention, an AFB4, AFB5 or SGT1bnucleic acid is used to identify whether a plant having anauxin-resistant phenotype has a mutation in endogenous AFB4, AFB5 orSGT1b or has a particular allele that causes auxin resistance.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. All publications cited herein are expressly incorporatedherein by reference for the purpose of describing and disclosingcompositions and methodologies that might be used in connection with theinvention. All cited patents, patent applications, and sequenceinformation in referenced public databases are also incorporated byreference.

Following are examples that illustrate procedures for practicingembodiments of the invention. These examples should not be construed aslimiting. All percentages are by weight and all solvent mixtureproportions are by volume unless otherwise noted.

Example 1 Identification and Genetic Characterization of MutantsResistant to DAS534

To identify mutants resistant to picolinate auxins, EMS-mutagenized M2seedlings were screened for resistance to DAS534, a 6-phenylpicolinateauxin (Compound 173 in WO 03/011853 A1). The seedlings were grown onagarose medium containing 10 nM DAS534. This concentration wassufficient to produce marked auxinic effects on the seedlings includingsignificant inhibition (60%) of root growth. From a total of 780,100 EMSM2 seedlings, 125 putative resistant mutants were identified by visualinspection. Seeds were recovered from 33 of these plants. Selection oflines for further study was based on the strength of resistance,elimination of potential siblings and the health and fertility of adultplants. To avoid recovering alleles of previously characterized mutants,we preferentially selected lines that showed low or no cross resistanceto 0.045 μM 2,4-D. Seven lines resistant to DAS534 had negligibleresistance to 2,4-D at this concentration (R009; R024; R045; R127; R051;R107; R118) and were further evaluated. One additional line (R090)showing robust resistance to both DAS534 and 2,4-D was also identified.

The mutants were crossed with wild-type Col-0 plants and the progenywere analyzed for resistance to DAS534. The F₁ plants that resulted fromthese crosses were all sensitive to DAS534 indicating that the mutationsin all of the resistant lines were recessive to the wild type allele. Todetermine the number of genetic loci identified in this screen,complementation tests were performed by crossing the herbicide-resistantmutants with each other and the known auxin-resistant mutant axr1-3 in apair-wise fashion. Analysis of the F1 progeny from these crosses bytesting for resistance to 10 nM DAS534 revealed that three distinctDAS534-resistant loci were present in the DAS534-resistant mutants.Complementation Group 1 contained the mutants R009, R024, R045 and R127,and complementation Group 2 contained the mutants R051, R107 and R118.All seven selectively-resistant mutant lines were phenotypically normalwhen grown on agarose medium or in soil and they had normal fertility.The seedlings exhibited normal gravitropism and had normal morphologyunder etiolating conditions. Group 2 mutants typically exhibitedslightly longer roots (−15%) than wild type plants when grown on agarosemedium lacking herbicide. Complementation Group 3 contained the mutantR090 which was resistant to both DAS534 and 2,4-D as well as the knownauxin-resistant mutant axr1-3. As the mutations in R090 and axr1-3appeared to be allelic, R090 was not examined further.

Example 2 Chemical Selectivity of Picolinate Auxin Resistant Mutants

The dose responses of root growth of the mutant lines treated with avariety of auxins were measured to define the level and chemicalspectrum of resistance in detail. Mutant lines from complementationGroups 1 and 2 had 6 to 8-fold resistance to DAS534 (FIG. 1A). Nosignificant differences in resistance between lines within eachcomplementation group were noted (data not shown). The mutants were verycross-resistant to the picolinate auxin herbicide picloram (FIG. 1B)showing 26 to 60-fold increases in GR₅₀ over that of wild type. Group 2mutants were slightly more resistant than Group 1 lines. In contrast tothe response to DAS534 and picloram, the lines from both complementationgroups showed negligible resistance to 2,4-D (FIG. 1C) and a slightincrease in sensitivity to IAA (FIG. 1D). The fold resistance levels ofthe mutants are summarized in Table 1.

Wild-type, Group 1 and Group 2 seedlings growing on 5 nM DAS534 areshown in FIG. 2. At this concentration of DAS534, wild-type plants showslight agravitopism, have elongated hypocotyls and are unable to fullyexpand their cotyledons (FIG. 2A and FIG. 2F) whereas hypocotylelongation and cotyledon expansion in the Group 1 mutant R127 and Group2 mutant R051 are unaffected by DAS534 (FIGS. 2B, 2D, 2G and 2I).

Four additional herbicidal compounds were also tested on the Group 1line, R127. R127 had 50-fold resistance to the picolinate auxinicherbicide clopyralid. However, it showed no resistance to the benzoateauxin dicamba or to a close analog of the aryloxyacetate auxin,fluoroxypyr or to 1-naphthylacetic acid (1-NAA). It also exhibited nodifference in response to the auxin transport inhibitor,napthylphthalamic acid (NPA).

In summary, the Group 1 and Group 2 mutants displayed clear chemicalselectivity in their resistance profiles toward the picolinate class ofauxin herbicides.

Example 3 Mapping and Identification of DAS534-Resistance Mutations

To identify the genes involved in this chemical selectivity, onemutation from each complementation group (R009 from Group 1 and R051from Group 2) was genetically mapped. To generate the mappingpopulation, a homozygous mutant M₃ line (from the Col-0 accession) wascrossed with a wild-type plant of the Ler accession and the resulting F₁plants were allowed to self-fertilize and produce F₂ seed. The F₂ seedwere germinated on solid medium containing a sublethal concentration ofDAS534. Plants resistant to the herbicide were removed from theherbicide-containing medium and allowed to recover on solid mediumlacking the herbicide for seven days and then transplanted to soil. Whenthe plants were at the rosette stage, a single leaf was removed andgenomic DNA was isolated and used in mapping experiments with moleculargenetic markers.

The mutation in R009 was mapped to a 200 kb interval around 105 cM onchromosome 5 between the markers C5_(—)104.6 and C5_(—)111 (FIG. 3).This interval contains 47 genes including At5g49980 annotated by theArabidopsis Information Resource as a homolog of TIR1. TIR1 is an F-boxprotein involved in 2,4-D and IAA-mediated SCF function and has recentlybeen shown to be a receptor for auxin (Dharmasiri et al., Nature.435(7041):441-445, 2005). tir1 mutants are resistant to 2,4-D and IAA(Ruegger et al., Genes Dev 12: 198-207, 1998). Because of this possiblelinkage to auxin function, At5g49980 was PCR amplified from genomic DNAfrom the four Group 1 mutant lines and Col-0 plants and compared by DNAsequence analysis. All four DAS534-resistant lines contained G to Amutations in At5g49980 relative to the Col-0 sequence resulting inchanges in the encoded polypeptide. The mutations in the deduced aminoacid sequences from R024 and R127 introduce stop codons at amino acidresidues 220 and 124, respectively. The mutations in R009 and R045introduce amino acid changes in the encoded polypeptide sequenceproducing an R to K mutation at position 609 in R009 and a C to Ymutation at position 451 in R045 (FIGS. 4A-4C).

There are six members of the F-box protein subclass that includes TIR1in the Arabidopsis genome (Gagne et al., Proc Natl Acad Sci USA 99:11519-11524, 2002). The three closest homologs have been named AFB 1, 2and 3 (Dharmasiri et al., Dev Cell. 9(1):109-119, 2005). Forconsistency, we have denoted the two remaining homologs (At4g24390 andAt5g49980) as AFB4 and AFB5 respectively. DNA and protein sequences forAFB4 are provided as SEQ ID NOS: 5 and 6, respectively. To confirm thatthe picolinate auxin resistant phenotypes in Group 1 mutants were causedby the identified lesions in AFB5, the auxin-resistance phenotype wascomplemented by transformation of the R127 mutant line with a constructcontaining a wild-type copy of AFB5 driven by the CsVMV promoter(Verdaguer et al., Plant Mol Biol 37: 1055-1067, 1998). T1 plantscontaining the transgene were selected, allowed to self-fertilize andset seed. T2 seeds that were segregating for the transgene weregerminated on medium containing 10 nM DAS534 and the seedlings werescored for sensitivity to the herbicide. Ten independent transformantswere identified that restored auxin sensitivity to the mutant (FIG. 2H).Some of these transformants appeared to be hypersensitive to DAS534because their roots were significantly shorter than those of wild-typeplants when grown on herbicide-containing medium. These data confirmthat the mutation in AFB5 is indeed responsible for the resistancephenotype in Group 1 mutants. As the DAS534-resistant mutants incomplementation Group 1 are resistant to picolinate auxins and not to2,4-D and all contain lesions in the leucine rich F-box protein AFB5,this protein appears to be specifically involved in mediating theeffects of picolinate auxins.

The R051 mutation was mapped to a 100 kb interval on chromosome 4 around36 cM between markers C4_(—)36 and C4_(—)37 (FIG. 3). This intervalcontains 17 genes including At4g11260 which encodes thetetratricopeptide-repeat containing protein, SGT1b (SEQ ID NO: 4). Amutation in SGT1b has recently been shown to enhance the level ofresistance to 2,4-D in the tir1 mutant background (Gray et al., PlantCell 15: 1310-1319, 2003). The deletion mutant line edm1 lacks sevengenes (At4g11220 through At4g11280) including SGT1b (Tor et al., PlantCell 14: 993-1003, 2002). We found that this deletion mutant had asimilar level of resistance to DAS534 as the Group 2 mutant R051 (FIG.5A). It was also very cross-resistant to picloram (20-fold, data notshown) similar to the Group 2 mutants. To determine if theDAS534-resistant mutants in complementation Group 2 contained mutationsin SGT1b, At4g11260 was PCR amplified from genomic DNA from all three ofthe mutants. Sequence analysis showed that all three mutants containedlesions producing alterations of the 3′ intron splice sites ofAt4g11260. Mutants R051 and R118 contained G to A transitions thatdisrupt the 3′ splice site of intron 7 whereas mutant R107 contained a Gto A transition that disrupts the 3′ splice site of intron 6 (FIG. 6).

To confirm that the mutations in At4g11260 were responsible for theDAS534-resistant phenotype, the mutant R051 was transformed with aconstruct containing the wild type At4g11260 gene driven by theconstitutive CsVMV promoter (Verdaguer et al., Plant Mol Biol 37:1055-1067, 1998). T2 seed were tested for DAS534 sensitivity. Using thesame process as was employed for identification of AFB5 transformants,ten independent transformants were identified that were sensitive toDAS534 confirming that the lesions in At4g11260 are responsible for theresistance to DAS534. An example of a R051 line containing the transgenethat restores sensitivity to DAS534 is shown in FIG. 2J.

Example 4 Comparison of the Phenotypes of Picolinate Auxin ResistantMutants with Other Auxin Resistant Mutants

The mutations presently identified in AFB5 and SGT1b conferringpicolinate-selective resistance implicate the SCF ubiquitin ligasecomplex in the molecular mode of action of these herbicides. Severalother mutants associated with this complex (e.g., tir1, axr1) or itsubiquitination targets (e.g., axr2) in the auxin response have beencharacterized and possess varying phenotypes and levels of resistance to2,4-D or IAA (Leyser et al., Nature 364: 161-164, 1993; Ruegger et al.,Genes Dev 12: 198-207, 1998; Nagpal et al., Plant Physiol 123: 563-573,2000). However, their chemical resistance profile to picolinate auxinshas not been previously established.

The mutant axr1-3 is defective in the rubinylation mechanism required toactivate SCF complexes. This mutant had a high level of resistance toDAS534 as well as to 2,4-D (FIGS. 5B and 5C) and so there was noindication of significant chemical selectivity between these two auxinclasses in this mutant. The axr1-3 mutant was also very resistant topicloram (160-fold). The relative degree of resistance of axr1-3 to thepicolinate auxins was higher than that of Group 1 and Group 2 mutantsindicating that this mutation has a more profound overall effect onauxinic responses. This is consistent with its general role in theactivation of SCF complexes (Schwechheimer et al., Plant Cell.14(10):2553-2563, 2002).

Mutants containing lesions in the F-box protein tir1 have significantlylower levels of resistance to 2,4-D than axr1 (FIG. 5C) suggesting thatadditional SCF complex components may also be involved in the 2,4-Dresponse (Ruegger et al., Genes Dev 12: 198-207, 1998; Dharmasiri etal., Nature 435:441-445, 2005). It was presently found that the modestlevel of resistance of tir1-1 to DAS534 was approximately equivalent tothat of 2,4-D thus the marked chemical selectivity observed with theGroup 1 and 2 mutants was not apparent with this mutant. The tir1-1mutant was slightly more resistant to picloram than DAS534 but thisresistance is considerably less than the resistance of either Group 1 orGroup 2 mutants to this compound. The resistance levels of tir1-1 andaxr1-3 to 2,4-D in our study were similar to those previously described(Estelle & Somerville, Mol Gen Genet. 206: 200-206, 1987; Ruegger etal., Genes Dev 12: 198-207, 1998), validating that the results from thesubject assay system are comparable to previous work using slightlydifferent assay methodologies.

AXR2 is one of the short-lived AUX/IAA transcriptional regulatorstargeted for SCF-mediated ubiquitination and mutations in these locihave been found to confer a dominant 2,4-D resistance phenotype (Wilsonet al., Mol Gen Genet. 222: 377-383, 1990; Nagpal et al., Plant Physiol123: 563-573, 2000). The roots of axr2 are agravitropic and elongatedrelative to wild type, corresponding to a loss of auxin regulation. Thelevel of resistance of axr2-1 to DAS534 was similar to that found with2,4-D and picloram and so there was no marked chemical specificity inresistance.

In summary, none of the three previously described auxin-resistantmutants that were tested showed the differential pattern of no or lowresistance to 2,4-D and high levels of resistance to picolinate auxinsthat we identified in the Group 1 and 2 mutants from our picolinateauxin resistance screen.

Example 5 Resistance Phenotype of AFB5 SGT1B Double Mutants

Mutations in either AFB5 or SGT1b cause similar levels of resistance toDAS534. Plants containing homozygous mutations in both AFB5 and SGT1bwere generated to determine if the level of resistance to DAS534 wassimilar or increased over that of the single mutant lines. Genomic DNAwas isolated from ninety-five F2 plants generated from a cross of R009and R051. The At5g49980 (AFB5) and At4g11260 (SGT1b) genes wereamplified by PCR and evaluated by DNA sequence analysis for mutations.Eight plants containing double mutations were identified and three ofthem were compared with the single mutant parental lines for resistanceto DAS534, picloram and 2,4-D. The single and double mutants exhibitedsimilar levels of resistance (5 to 10-fold, data not shown), thus theresistance mechanisms in afb5 and sgt1b are not additive. The doublemutants also exhibited no obvious deleterious phenotype in growth orfertility, similar to the single mutants. These data therefore suggestthat AFB5 and SGT1b are involved in the same response pathway to DAS534.

Example 6 Functional Assay for AFB5

A biochemical pull-down assay to monitor the picolinate auxin-responseof AFB5 is constructed using the methods described in Dharmasiri et al.(Cum Biol. 13:1418-1422, 2003). However, plant extracts for the assaysare made from transgenic Arabidopsis afb5 plants expressing myc-taggedAFB5 rather than tir1 plants expressing TIR1-myc. The recovery ofAFB5-myc is monitored by immunoblotting with anti-c-myc antibody. Testcompounds are added into the assay mixture and the reduction in recoveryof AFB5-Myc is monitored.

Example 7 Ligand-Binding Assay for AFB5

A biochemical assay to monitor ligand-binding by AFB5 is constructedusing the methods described in Dharmasiri et al. (Nature 435:441-445,2005) and Kepinski and Leyser (Nature, 435:446-451, 2005). However,plant extracts for the assays are made from transgenic Arabidopsis afb5plants expressing myc-tagged AFB5 rather than tir1 plants expressingTIR1-myc. Also [³H]-picloram, [³H]DAS534 or other tritiated picolinateauxin is used as the radioligand instead of [³H]IAA or [³H]2,4-D. Testcompounds are added into the assay mixture and the displacement ofbinding of tritiated picolinate auxin is monitored.

Example 8 Sequence Comparisons and Identification of Additional Homologs

BLAST searches of the NCBI databases using the AtAFB5 sequenceidentified six AFBs in the Arabidopsis genome (AtTIR1, AtAFB1-5), 4 AFBhomologs from the rice (Oryza sativa) genome, and a homolog of AFB5 fromPopulus. All of these sequences were aligned using CLUSTALW within theAlignX module of Vector NTI.

From this CLUSTAL alignment, a phylogenetic analysis was performed usingthe neighbor-joining method in MEGA3.1 (S Kumar et al. (2004) Briefingsin Bioinformatics 5:150-163) (% bootstrap values from 1000 iterationsare shown). As illustrated in FIG. 7, this shows that AFB5, AFB4, and 4other related AFBs (Os NP 912552, Os XP 507533, Pt AAK16647) aredistinct from previously characterized TIR1 and 5 TIR1-related AFBs(AtAFB14g03190, AtTIR1At3g62980, Os XP 493919, Os CA D40545, AtAFB23g26810, and AtAFB3 1g12820). Members of the TIR1 group share at least42% amino acid identity with all other members of that group.

Across the entire proteins, AFB4 and AFB5 share about 80% amino acididentity with each other. Members of the AFB4 and AFB5 group all have atleast 54% identity with all other members of the group. The proteinsequence from Populus has about 70% identity with AFB4 and about 73%identity with AFB5. All members of this group are considered to bewithin, and usable according to, the subject invention. The mRNA andprotein sequences, respectively, of the four AFB5 homologs are attachedas SEQ ID NOS: 7 and 8 (Oryza sativa (rice) (Genbank entry gi:34902411),SEQ ID NOS: 9 and 10 (Oryza sativa (rice) (Genbank entry gi:51979369),and SEQ ID NOS:11 and 12 (from Populus).

Example 9 Resistance of AFB5 Mutant to Foliar Application of PicolinateAuxin Herbicides

Arabidopsis seedlings were grown for two weeks in a growth chamber (23°C.; continuous light at 120-150 μE m⁻² sec⁻¹), then taken to thegreenhouse for three days after which plants were sprayed with herbicideusing a track-sprayer to deliver the appropriate rate. Plants were thengrown in the greenhouse for 12 days (22° C. under supplemented lightwith a 14 h: 10 h light-dark cycle) prior to photography and evaluation.A foliar spray application of picloram or aminopyralid at 200 g/ha onwild type Col-0 Arabidopsis plants growing in the greenhouse producedprofound morphological effects and severely inhibited plant growth,whereas the applications had minimal effect on the Group 1 mutant R009(FIG. 8A-8C). This is in contrast to the effect of 2,4-D which inducesauxinic symptoms and growth reduction to a similar extent on both wildtype and mutant plants at 50 g/ha. Thus the chemical selectivity ofresistance seen in seedling plate assays is maintained in adult plantswith foliar exposure to the auxin herbicides.

ADDITIONAL REFERENCES

-   Balko et al. (2003) Preparation of 6-aryl-4-aminopicolinic acids as    herbicides with excellent crop selectivity. 2002-US24120: 84-   Klein et al. (1987) High-velocity microprojectiles for delivering    nucleic acids into living cells. Nature (London, United Kingdom)    327: 70-73-   Smith et al. (1988) Antisense RNA inhibition of polygalacturonase    gene expression in transgenic tomatoes. Nature (London, United    Kingdom) 334: 724-726

We claim:
 1. A method of identifying an herbicidal compound that bindswith an AFB4 protein, the AFB4 protein comprising a polypeptidecomprising an amino acid sequence having at least 95% amino acidsequence identity with SEQ ID NO: 6, the method comprising: contactingthe AFB4 protein with a test compound; assaying the AFB4 protein forbound compound; and screening the compound for herbicidal activity. 2.The method of claim 1, wherein the AFB4 protein comprises a polypeptidecomprising an amino acid sequence set forth as SEQ ID NO:
 6. 3. Themethod of claim 1, wherein the AFB4 protein consists of a polypeptidehaving an amino acid sequence set forth as SEQ ID NO:
 6. 4. The methodof claim 1, wherein the herbicidal compound is an auxin herbicidalcompound.
 5. The method of claim 1, wherein the herbicidal compound is apicolinate auxin herbicidal compound.